The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.

The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 263-321) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008.

With the development of Highly Active Anti-Retroviral Therapy (HAART), Human Immunodeficiency Virus (HIV) infection has evolved from a fatal disease to a chronic condition with increased risk for non-infectious comorbidities, including reduced bone density. Bone density is maintained through the coupled activities of osteoblast matrix deposition and osteoclast resorption; while uncoupling this process can result in bone loss and increased fracture risk. CD4+ T cells are critical in regulating the activity of these cells. Relevant to this thesis, studies have shown that HAART treated patients experience higher levels of immune activation; possibly contributing to the observed bone loss. If osteoimmune dysregulation does occur, there is a need to develop therapeutics that target this process, especially in the context of HIV infection. To evaluate osteoblast differentiation, we developed a high-throughput screening method to identify osteo-regulatory compounds. By screening over 5,000 compounds, we identified 18 that robustly induced osteoblast differentiation, using a mouse mesenchymal stem cell. We validated two of these compounds, rapamycin and FK-506, which are known immunosuppressants. Secondly, we addressed the role of activated CD4+T cells and HIV-infected T cells in osteogenesis. We found that supernatants from activated T cells potently inhibit osteoblast differentiation. However, when osteoblasts were co-cultured with HIV-infected T cells, differentiation was inhibited regardless of activation status, suggesting intrinsic differences between HIV infected and uninfected T cells. Finally, to prevent the inhibition of osteogenesis by activated T cells, we evaluated rapamycin, our pro-osteogenic and T cell activation antagonist, as well as the novel compound JQ1, an inflammatory inhibitor that targets bromodomain-containing proteins. Both rapamycin and JQ1 efficiently blocked the cytotoxic effects of supernatants from non-infected activated T cells on osteoblasts, whereas only rapamycin prevented inhibition in the co-culture model. In contrast, neither rapamycin nor JQ1 were able to prevent inhibition by HIV infected, activated T cells. This suggests that HIV exacerbates the negative effects of T cell activation on osteoblastogenesis. These data support a mechanism for HIV infection and T cell activation mediating bone loss.

MTor est une protéine centrale de la voie de signalisation PI3K/AKT/mTor et est impliquée dans la croissance, la prolifération et la survie cellulaire. Cette protéine joue un rôle majeur particulièrement dans la prolifération tumorale. Ainsi des inhibiteurs de mTor ont été développés à partir des années 1980, notamment la rapamycine, et deux sont utilisés actuellement en clinique: l’évérolimus et le temsirolimus. Ces deux inhibiteurs font partie des différentes possibilités thérapeutiques dans les cancers du sein et du rein métastatiques. Les patients traités présentent des réponses variées avec des cas de patients très bons répondeurs et des cas de patients résistants d'emblée ou après exposition au traitement. Face à cette notion de résistance, nous avons voulu caractériser les mécanismes d’adaptation de la cellule tumorale après exposition aux anti-mtor. L’analyse de l’expression génomique de huit lignées cellulaires tumorales variées après traitement par la rapamycine a montré une modulation de l’expression de certains gènes dont celle de la pseudokinase TRIB3, diminuée dans toutes les lignées. Cette donnée in vitro est aussi retrouvée chez les patients. En effet après traitement par évérolimus, nous avons pu observer une diminution de l’expression du gène TRIB3 au niveau sanguin. Nous avons donc cherché à comprendre le rôle de cette pseudokinase dans la réponse aux anti-mTor. Nos résultats mettent en évidence, d’une part, que la rapamycine régule l’expression de TRIB3 en agissant sur son promoteur via une interaction avec GCF2. D’autre part, l’hyperexpression de TRIB3 limite les effets anti-tumoraux de la rapamycine dans différentes lignées cellulaires tumorales. Pour étudier plus en détails ce mécanisme, nous avons cherché à déterminer les partenaires de TRIB3. Par des approches de protéomique haut-débit, nous avons mis en évidence un lien avec des protéines impliquées dans l’épissage. Ainsi la rapamycine semble inhiber la machinerie d’épissage via la diminution d’expression de TRIB3. Ce travail relève l’intérêt de TRIB3 dans la réponse aux anti-mTor comme un potentiel biomarqueur et illustre également le mode d’action de la rapamycine. / MTor is a central protein of the PI3K/AKT/mTor signaling pathway and is involved in growth, proliferation and cell survival. This protein plays a major role particularly in tumor proliferation. Thus, mTor inhibitors have been developed since the 1980s, including rapamycin, and two are currently used in daily practice: everolimus and temsirolimus. These two inhibitors are part of the different therapeutic possibilities in metastatic breast and kidney cancers. Patients treated present different responses with cases of very good responders and cases of resistant patients either immediately or after exposure to treatment. According to the concept of resistance, we wanted to characterize the mechanisms of adaptation of the tumor cell after exposure to mTor inhibitors. Analysis of the genomic expression of eight variant tumor cell lines after treatment with rapamycin showed a modulation of the expression of different genes including the pseudokinase TRIB3, decreased in all the lines. This in vitro data is also found in patients. Indeed, after treatment with everolimus, we observed a decreased expression of the TRIB3 gene in blood. We therefore sought to understand the role of this pseudokinase in the response to mTor inhibitors. Our results show, that rapamycin regulates the expression of TRIB3 through a GCF2-dependent inhibition of promoter activity. Moreover, TRIB3 overexpression limits the anti-tumor effects of rapamycin in different tumor cell lines. To investigate this mechanism, we sought to identify TRIB3 partners. By high-throughput proteomic approaches, we have demonstrated a link with proteins involved in splicing. Thus, rapamycin appears to inhibit splicing machinery with the decreased expression of TRIB3. This work highlights the interest of TRIB3 in the response to mTor inhibitors as a potential biomarker and investigates also how rapamycin acts on cells.

Mitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonuclease-deficient MEFs were more sensitive to several mitochondrial inhibitors and showed increased reactive oxygen species (ROS) production under standard conditions. When cultured in pyruvate-containing media, POLG exonuclease-deficient MEFs showed decreased oxygen consumption compared to controls. Increased AMP-activated protein kinase (AMPK) signaling and decreased mammalian target of rapamycin (mTOR) signaling delayed aging and influenced mitochondrial function. Therefore, the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, or rapamycin, an mTOR inhibitor, on measures of mitochondrial function were determined. Rapamycin treatment transiently increased respiration only in WT MEFs and, under most conditions, increased ATP levels. Short term AICAR treatment transiently increased ROS production and, under most conditions, decreased ATP levels. Chronic AICAR treatment decreased respiration and ROS production in WT MEFs. These results demonstrate the context-dependent effects of AICAR and rapamycin on mitochondrial function.

No description available.

No description available.

No description available.